CN117886952B - Antibody and matched pig sperm freezing kit thereof - Google Patents
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- Peptides Or Proteins (AREA)
Abstract
The invention provides an antibody and a matched porcine sperm freezing kit thereof, and the antibody can be used for detecting whether a sample contains common epidemic strains of swine fever virus, porcine circovirus and porcine reproductive and respiratory syndrome virus. Therefore, the kit can be rapidly applied to the detection process of frozen semen of an external pig, can conveniently, rapidly and accurately detect the three pathogens, effectively block the vertical transmission of the pathogens and prevent diseases. The detection reagent can be matched with pig semen diluent for use, ensures the purity of the product, can be widely applied to semen preservation and operation in pig farms, and can be used for adjusting the pig semen market, enhancing quarantine, eliminating healthy pigs with toxicity, purifying epidemic diseases of the breeding pigs and achieving good economic and social benefits.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an antibody and a matched porcine semen freezing kit thereof, in particular to an antibody for rapidly detecting swine fever virus, porcine circovirus and porcine reproductive and respiratory syndrome virus in porcine semen and application of the antibody in cryopreservation of porcine semen.
Background
Along with the development of scientific technology, the living standard of people is continuously improved, the requirements on livestock products are also higher and higher, the demand is also higher and higher, the pig industry plays an important role in the livestock industry system, along with the continuous improvement of the intensification and standardization degree of the pig industry, the artificial insemination technology is also continuously developed and perfected and widely applied, and becomes the most direct and effective way for improving pig breeds and breeding. In order to improve the quality of local pigs, the direct import of frozen boar semen from abroad and even abroad is also a new introduction means, but the problem of carrying poison with healthy boar semen is increasingly complicated and serious.
Studies have shown that viruses such as classical swine fever virus (CLASSICAL SWINE FEVER virus, CSFV), porcine circovirus (Porcine circovirus, PCV), porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV) are the most commonly detected pathogens in the semen of introduced boars, and are important viral pathogens causing porcine reproductive disorders. The clinical symptoms of the virus are mainly represented by abortion, premature birth, infertility, stillbirth, mummy embryo, weak baby and the like of sick pregnant sows, and can also cause morbidity or mortality of suckling piglets and nursery pigs. The pathogens can be horizontally transmitted and can be vertically transmitted through the placenta of the sow and the semen of the boar. It can be seen that boar semen is an important medium for the transmission of pathogenic agents of reproductive disorders. With the development of molecular biology technology, a rapid and efficient molecular biology method becomes an important detection means for the propagation disorder diseases.
However, the common detection means known in the prior art often aims at samples with larger viral loads such as serum and tissue samples of pigs to be detected, and the detection reagent and the detection means are not specially aimed at the porcine sperm. Moreover, although the conventional RT-PCR method can detect viral genome and can make diagnosis relatively accurately, the method has the phenomena of omission and cross contamination, complicated operation and instrument are required, and multiple pathogens are difficult to detect in a rapid and large-scale manner, so that multiple tests are often required to obtain corresponding results, time and labor are wasted, and the key is to waste the originally precious frozen sperm sample.
Therefore, there is an urgent need for a detection reagent and a detection method thereof that can detect several viruses in frozen semen of pigs at the same time.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a novel antibody capable of identifying epidemic strains of swine fever virus, porcine circovirus and porcine reproductive and respiratory syndrome virus simultaneously.
Specifically, the invention firstly provides an antigen epitope obtained by fusion expression of the strain optimization to form a novel antigen epitope fusion protein CPP, and the amino acid sequence of the antigen epitope fusion protein is shown as a sequence table SEQ ID No. 1.
The invention also provides a nano antibody CPP-Nb1 for specifically recognizing the epitope fusion protein CPP, wherein the amino acid sequence of the nano antibody CPP-Nb1 is shown as SEQ ID NO.2
The invention also provides a nucleic acid molecule for encoding the nano antibody CPP-Nb1, and the nucleotide sequence is shown as SEQ ID NO. 3.
The invention also provides a fusion protein containing the nano-antibody CPP-Nb1 and a marker locus.
The invention also provides a recombinant plasmid for expressing the fusion protein, and a basic vector of the recombinant plasmid comprises a prokaryotic expression vector.
The invention also provides a preparation method of the fusion protein, which comprises the following steps: transferring the recombinant plasmid into competent cells, performing induction expression by using IPTG, collecting inclusion body proteins, and performing labeling of the labeling sites after purification.
The invention also provides application of the nano-antibody CPP-Nb1, the fusion protein or the recombinant plasmid in preparation of reagents for detecting the three pathogenic antibodies in the porcine semen.
The invention also provides a detection kit for simultaneously detecting epidemic strains of swine fever virus, porcine circovirus and porcine reproductive and respiratory syndrome virus in porcine semen. Further, the detection method of the kit comprises an enzyme-linked immunosorbent assay, a chemiluminescent detection method, a western blot detection method or an immunohistochemical method.
Further, the invention provides a pig semen freezing kit, which comprises a detection component and a freezing component, wherein the detection component comprises an epitope fusion protein CPP and/or a nanobody CPP-Nb1, and the freezing component comprises:
base dilution:
BTS:37g/L glucose, 1.25g/L EDTA disodium, 6g/L sodium citrate, 0.75g/L potassium chloride, 1.25g/L sodium bicarbonate, 300mg/L gentamicin.
TCG:5.5g glucose, 7.4g sodium citrate, 12.0g tris, 50 ten thousand units of penicillin and streptomycin each, 100mL egg yolk, and 500mL distilled water.
And (3) liquid I: the BTS and the TCG are compatible according to the volume ratio of 1:1.
II, liquid: and adding 6% glycerol into the solution I.
And (3) a dilution step: the total amount of dilution required is calculated as the final concentration of not less than 4X 10 8/mL. Diluting fresh semen with half volume of solution I, balancing at 4deg.C for 1.5 hr, adding solution II precooled at 4deg.C, subpackaging with thin tube, and freezing.
Advantageous effects
The antibody disclosed by the invention can be used for detecting whether a sample contains common epidemic strains of swine fever virus, porcine circovirus and porcine reproductive and respiratory syndrome virus. Therefore, the kit can be rapidly applied to the detection process of frozen semen of an external pig, can conveniently, rapidly and accurately detect the three pathogens, effectively block the vertical transmission of the pathogens and prevent diseases. The detection reagent can be matched with pig semen diluent for use, ensures the purity of the product, can be widely applied to semen preservation and operation in pig farms, and can be used for adjusting the pig semen market, enhancing quarantine, eliminating healthy pigs with toxicity, purifying epidemic diseases of the breeding pigs and achieving good economic and social benefits.
Drawings
FIG. 1 is a schematic diagram showing three-dimensional structure prediction of an epitope fusion protein CPP obtained by fusion expression of classical swine fever virus, porcine circovirus type 2 and optimization of epidemic strains of porcine reproductive and respiratory syndrome virus.
Detailed Description
The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The invention will be further illustrated by the following examples
Example 1 screening and analysis of dominant epitopes of classical swine fever Virus, porcine circovirus, porcine reproductive and respiratory syndrome Virus epidemic strains
Since the three viruses, namely, the hog cholera virus, the porcine circovirus and the porcine reproductive and respiratory syndrome virus, have a plurality of encoded proteins and complex infection host mechanisms, the current research on the three pathogenic antigens is mostly stopped at the protein level, and only a few epitopes of key antigens are deeply analyzed by researchers. Therefore, the applicant has recorded 384 CSFV epitopes reported in 58 papers and 532 porcine circovirus type 2 epitopes reported in 167 papers in the IEDB database by 8 months of 2023, and has performed a finishing analysis on 309 PRRSV epitopes reported in 212 papers, selected the main antigen proteins of each strain which are popular in recent years and the variation of the epitopes, and combined with the characteristics of the porcine semen, and performed a screening analysis on the B cell epitopes, T cell epitopes and MHC molecule ligands thereof, thereby finally obtaining candidate sets of the epitopes shown in table 1.
TABLE 1 epitope for different viruses
The analysis and processing of the antigen epitopes are carried out by using bioinformatics, the combination of specific antigen epitopes is selected from the antigen epitopes, various antigen proteins are covered as much as possible to achieve the effect of wide recognition, meanwhile, individual epitopes in the selected antigen epitopes are changed and modified by using 3D modeling, connection is carried out by using rigid and flexible linker (figure 1), and the specific amino acid sequence of the antigen epitope fusion protein is finally obtained:
TMSAHENLRGGSSSMVKEALAKYGSGSERLYELTYDPYVNYMGSSIDDFGGGSSSGGGSSSPLCPTRQAHAEILEV VLQDIDHFCNDSTAPQGSGSTQHTVRLISATASPSAGGSSELNGTDWLTQKFDWAGSGSRKVMTSKKPHSRYFTPWNNSN INNFTLFEQERS(SEQ ID NO.1).
the identification of the serum sample of the fusion protein and the epidemic strain is verified by using a Dot-blot method, and the method specifically comprises the following steps:
(1) Firstly, an amino group on carrier protein BSA is activated by utilizing a heterobifunctional crosslinking agent Sulfo-SMCC, and is connected with a sulfhydryl group of cysteine at the N end of the polypeptide to form coupling of the polypeptide and the carrier protein, and the concentration of the coupled polypeptide is regulated by using physiological saline for standby at-20 ℃.
(2) Activating PVDF membrane with anhydrous methanol, balancing with buffer solution, printing coupled polypeptide on PVDF membrane at 2 μg/dot, and using carrier protein (BSA) as negative control;
(3) Closing: after the spots of the coupled polypeptide are naturally dried, putting the membrane into 5% skimmed milk solution, sealing the membrane for 1.5 hours at 37 ℃ by a shaking table, pouring off sealing liquid, and washing the membrane with PBST washing liquid for 3 times for 5 minutes each time;
(4) An antibody: immersing the membrane into positive serum and negative serum control of each epidemic virus strain diluted by 1:200, incubating for 1-1.5h at 37 ℃ in a shaking table, and washing 3 times with PBST washing liquid for 5min each time;
(5) And (2) secondary antibody: immersing the membrane in a sheep anti-pig IgG-HRP solution diluted by 1:3000, and washing with PBST after shaking table incubation at 37 ℃ for 1 h;
(6) Color development: mixing the developing solution A and the developing solution B in equal quantity, and preparing for use. The sample film was subjected to chemiluminescence and the results recorded.
The results are shown in Table 2, and although the positive results are strong or weak as seen in the lattice diagram, the fusion protein can perform positive reaction with almost all epidemic strains as a whole, and can be used as a target for conventional virus screening.
Table 2 results of serum reaction of fusion proteins and epidemic strains
Example 2: immune panning process of natural single domain antibody aiming at antigen epitope fusion protein
(1) Amplifying the established natural single domain antibody phage library: adding 2 XYT culture medium into 100 mu L of glycerol bacteria library, adding 20MOI auxiliary phage when OD600 = 0.5, standing for 30min, centrifuging, re-suspending the precipitate with 2 XYT culture medium, culturing for 1h, culturing with antibiotic for 16h, centrifuging, and re-suspending the supernatant with pre-cooled PEG-NaCl (1/4 volume) solution and 1mL PBS solution to obtain amplified single domain antibody library;
(2) Immune tube panning: coating 50 mug/tube of the fusion protein (full-length expression of Huada gene (Shanghai), carrying biotin tag and purification) on an immune tube overnight, removing coating liquid, washing 3 times, blocking 2h with 2mL BSA (1%), washing 3 times with PBST, adding 100 mug (1) of the single domain antibody library amplified in the step (1) as primary antibody, reacting for 2h at 37 ℃, washing 3 times with PBST, eluting with Glycine-HCL (PH 2.2), and regulating the pH of eluent to 7.4 with Tris-HCL to obtain a panned first round natural single domain antibody library;
(3) Amplifying the round 1 natural single domain antibody library obtained in the step (2) according to the step (1) to obtain a round 1 natural single domain antibody re-suspension library, repeating the step (2) of immune tube panning, wherein only 100 mu L of amplified round 1 natural single domain antibody re-suspension library is added to the primary antibody, and finally obtaining a panned round 2 natural single domain antibody library;
(4) Amplifying the round 2 natural single domain antibody library obtained in the step (3) according to the step (1) to obtain a round 2 natural single domain antibody re-suspension library, repeating the step (2) of immune tube panning, wherein 100 mu L of amplified round 2 natural single domain antibody re-suspension library is added to the primary antibody, and finally obtaining the panned round 3 natural single domain antibody library.
Example 3: ELISA identification of individual clones
(1) Panning of single positive clones for antibody expression: the 3 rd round of natural single domain antibody library panned in the above steps is inoculated in a2 XYT culture medium, 20MOI helper phage is added when OD 600nm = 0.5, the mixture is left for 30min, the precipitate is resuspended in the 2 XYT culture medium after centrifugation, and then cultured for 1h, and then coated on a2 XYT plate containing antibiotics, cultured overnight, 40 single colonies are selected the next day to be inoculated in the 2 XYT culture medium, 20MOI helper phage is added when OD600 = 0.5, the mixture is left for 30min, the precipitate is resuspended in the 2 XYT culture medium after centrifugation, and then cultured again, and IPTG is added for induced expression for 8h. (2) ELISA identification: 100 mu L/well of the fusion protein (Hua Dayin gene (Shanghai) full-length expression, carrying biotin tag and purification, concentration 1 ng/. Mu.L) was coated on ELISA plate overnight, washed 3 times after removing coating liquid, blocked with 200 mu L/well BSA (3%) for 2h, washed 3 times with PBST, added with 100 mu L/well (1) of single domain antibody library amplified in each step as primary antibody (library construction vector M13), acted 2h at 37 ℃, washed 3 times with PBST, added with M13-HRP, and after termination, OD450nm value was detected, and the result revealed: positive was judged to be 3-fold higher than the OD450nm value of the control group.
Example 4: sequencing identification of Positive clones
The clones detected positive by ELISA in example 3 were extracted with plasmids corresponding to the respective bacterial solutions, and sequenced using the universal primers of the plasmid vectors. And analyzing the gene sequence of each clone strain according to sequence comparison software MEGA 6.0, taking the strains with the same CDR1, CDR2 and CDR3 sequences as the same clone strain and the strains with different sequences as different clone strains, and finally obtaining the specific single-domain antibody sequence of 4 strains aiming at the antigen epitope fusion protein. Among them, the best strain was selected, and the antibody had a nucleotide sequence SEQ ID NO：2(GATGTGCAGCTGCAGGAAAGCCAGCCGGGCAGCGGCCTGGTGGGCGGCAGCCGCCTGAGCTGCGCGGGCAGCCAT CGCAGCCTGACCTTTTATGCGATTGGCTGGTTTCGCGCGCCGCCGCGCAAAGAACCGGAAAGCGTGAGCTGCATTACCCCGGGCCCGCATACCCAGATTCTGATTGATAGCCATAAAGGCCGCTTTGATATTAGCCGCGATGAAGCGAAAGCGACCGTGTATCTGCAGATGAACAGCCTGGAACCGGAAGATACCGGCCATTATGCGTGCAGCGCGGATGAAAACGCGCCGTATCATGCGGTGCATGGCCAGAGCGAAATGTATGATTATTGGGAACAGGAAACCGCGGTGACCGTGAGCAGC), and a CDR1-CDR2-CDR3 region shown as an underlined portion of SEQ ID NO：3(DVQLQESQPGSGLVGGSRLSCAGSHRSLTFYAIGWFRAPPRKEPESVSCITPGPHTQILIDSHKGRFDISRDEAKATV YLQMNSLEPEDTGHYACSADENAPYHAVHGQSEMYDYWEQETAVTVSS)., and constituted the VHH chain of the subject.
Example 5 detection of Virus in porcine semen
Boar semen samples were from the boars of 12 pig farms in Sichuan, 282 parts (heads).
Base dilution:
BTS:37g/L glucose, 1.25g/L EDTA disodium, 6g/L sodium citrate, 0.75g/L potassium chloride, 1.25g/L sodium bicarbonate, 300mg/L gentamicin.
TCG:5.5g glucose, 7.4g sodium citrate, 12.0g tris, 50 ten thousand units of penicillin and streptomycin each, 100mL egg yolk, and 500mL distilled water.
And (3) liquid I: the BTS and the TCG are compatible according to the volume ratio of 1:1.
II, liquid: and adding 6% glycerol into the solution I.
And (3) a dilution step: the total amount of dilution required is calculated as the final concentration of not less than 4X 10 8/mL. Diluting fresh semen with half volume of solution I, balancing at 4deg.C for 1.5 hr, adding solution II precooled at 4deg.C, subpackaging with thin tube, and freezing. And respectively taking a sample before freezing and a sample after thawing after freezing and preserving for 120 hours to detect the virus antibody.
Coating: diluting the epitope fusion protein to 2 mu g/mL by using 0.05mol/L Na 2CO3-NaHCO3 buffer solution with the pH value of 9.6, adding 100 mu L of the buffer solution into each hole, and adsorbing for 14 hours at the temperature of 4 ℃;
After coating, discarding the liquid in the holes, adding 250 mu L of PBS (phosphate buffered saline) washing liquid containing 0.05% Tween-20 into each hole, washing for 3 times, and finally beating to dryness;
Closing: 200 mu L of PBS blocking solution containing 5% BSA and 0.05% Tween-20 is added into each hole, and the mixture is blocked for 1h at 37 ℃;
After the sealing is finished, discarding the liquid in the holes, adding 250 mu L of washing liquid into each hole, washing for 3 times, and finally beating to dryness;
incubation resistance: diluting the semen sample to be detected by using a sample diluent according to the ratio of 1:100, adding 100 mu L of the sample diluent into each hole, and reacting for 60min at 37 ℃;
discarding the liquid in the holes, adding 250 mu L of washing liquid into each hole, washing for 5 times, and finally beating to dryness;
secondary antibody incubation: diluting the enzyme-labeled secondary antibody with an antibody diluent according to the ratio of 1:10000, adding 100 mu L of the enzyme-labeled secondary antibody into each hole, and reacting for 60min at 37 ℃;
discarding the liquid in the holes, adding 250 mu L of washing liquid into each hole, washing for 5 times, and finally beating to dryness;
Color reading: 100 mu L of single-component TMB substrate developing solution is added into each hole, the reaction is carried out for 15min at 37 ℃ in a dark place, 100 mu L/Kong Zhongzhi of solution is added, an OD 450nm value is read by an enzyme labeling instrument, a P/N value is calculated, a sample with the P/N value more than or equal to 2.1 is judged as positive, and a sample with the P/N value less than 2.1 is judged as negative.
Wherein, the sealing liquid: 5% of skimmed milk powder;
W-TMB color development liquid: a biological organism: c510025;
coating liquid: 0.05M carbonate buffer, ph=9.6;
Washing liquid: 1 XPBST;
stop solution: 1mol/LHCL;
enzyme-labeled antibody: the working concentration of the HPR marked goat anti-pig secondary antibody is diluted by the antibody protection liquid.
Meanwhile, PCR detection is carried out as a reference, and total DNA in semen is extracted by using a DNA extraction kit (Beijing days is an age science and technology Co., ltd.).
The PCR reaction system was 2X Pro Taq MasterMix (Ai Kerui G11112) 10. Mu.L, 1. Mu.L of the upstream primer (PrimerF.), 1. Mu. L, RNase to FREE WATER. Mu.L of the downstream primer (PrimerR.), and 3. Mu.L of the Template (Template). The reaction procedure is: pre-denaturation at 95 ℃ for 30s, denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, setting 35 reaction cycles, and final extension at 72 ℃ for 5min;
the primers used are shown in Table 3:
TABLE 3 PCR primer sequences involved in this study
From the results (Table 4), it can be seen that the detection result obtained by using the epitope fusion protein can replace the traditional PCR detection to a certain extent, the ELISA method is simple and convenient to operate, can be completed without complex instruments, meets the high-speed and high-flux detection requirements in a large-scale pig farm, and has good application prospect.
TABLE 4 detection results of different detection means
And the subsequent experiments further prove that the detection result is not changed before and after freezing, and the freezing operation is proved to have no adverse effect on the detection of the viruses, so that the detection method or the kit and the cryopreservation kit can be suggested to be sold in a binding way, the detection of the conventional viruses is carried out, and the preservation of the seed extract is carried out according to the corresponding detection result.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
The foregoing is merely illustrative embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily think about variations or substitutions within the technical scope of the present invention, and the invention should be covered. Therefore, the protection scope of the invention is subject to the protection scope of the claims.
Claims (4)
1. An epitope fusion protein containing epidemic strains of classical swine fever virus, porcine circovirus and porcine reproductive and respiratory syndrome virus is characterized in that the antigen epitopes of the three viruses are optimized to form a novel epitope fusion protein CPP, and the amino acid sequence of the epitope fusion protein is shown as a sequence table SEQ ID No. 1.
2. A nanobody CPP-Nb1 specifically recognizing the epitope fusion protein CPP of claim 1, wherein the amino acid sequence of the nanobody CPP-Nb1 is shown in SEQ ID No. 3.
3. The application of the epitope fusion protein of claim 1 and the nanobody CPP-Nb1 of claim 2 in preparing reagents for detecting swine fever virus, porcine circovirus and porcine reproductive and respiratory syndrome virus in porcine semen.
4. A pig semen freezing kit comprising a detection component and a freezing component, wherein the detection component comprises the epitope fusion protein of claim 1 and/or the nanobody CPP-Nb1 of claim 2, and the freezing component comprises:
base dilution:
BTS:37g/L glucose, 1.25g/L EDTA disodium, 6g/L sodium citrate, 0.75g/L potassium chloride, 1.25g/L sodium bicarbonate, 300mg/L gentamicin;
TCG:5.5g glucose, 7.4g sodium citrate, 12.0g tris, 50 ten thousand units of penicillin and streptomycin each, 100mL egg yolk, 500mL distilled water;
And (3) liquid I: the BTS and the TCG are compatible according to the volume ratio of 1:1;
II, liquid: adding glycerol with the final concentration of 6% into the solution I;
And (3) a dilution step: calculating the total amount of required diluent according to the final concentration of not less than 4X 10 8/mL; diluting fresh semen with half volume of solution I, balancing at 4deg.C for 1.5 hr, adding solution II precooled at 4deg.C, subpackaging with thin tube, and freezing.
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